Abnormal activation of hypoxia-inducible factor-1 (HIF-1

FKHRL1 (also calledFOXO3a

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor, or erythropoietin (EPO) for growth and survival. We investigated the effect of thrombopoietin (TPO), the ligand for the receptor encoded by c-mpl proto-oncogene, on the proliferation and differentiation of UT-7 and its sublines. We found that UT-7/GM, which is a subline of UT-7, but neither UT-7 nor UT-7/EPO, can proliferate in response to TPO. The subline, UT-7/TPO, was established from UT-7/GM by culture at lower concentrations of TPO. UT-7/TPO cells had morphologically mature megakaryocytic characteristics such as developed demarcation membrane in the cytoplasm and multinucleated appearance. This was also confirmed by the high expression of platelet factor-4 and glycoprotein IIb at the mRNA levels and by the high level of DNA content. UT-7/TPO can be maintained by TPO alone, with a doubling time of 24 hours in log growth phase. In the absence of TPO, the majority of the cells died within a few days. Thus, UT-7/TPO has an absolute dependence on TPO for growth and survival and has mature megakaryocytic features. The mRNA for c-mpl was detected in UT-7/TPO and, to a lesser degree, in UT-7/GM. The mRNA level of NF- E2 p45, reported to be an erythroid-specific transcription factor, was upregulated in UT-7/TPO, whereas it was down-regulated in the erythroid subline, UT-7/EPO. There were no significant differences in GATA-1 and GATA-2 mRNA levels among UT-7 and its sublines. Not only EPO but also TPO induced the tyrosine phosphorylation of JAK2 tyrosine kinase and STAT5-related protein. These findings indicate that UT-7/TPO would be a useful model with which to analyze the gene regulation of megakaryocytic maturation- associated proteins and to study the specific actions of TPO.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF ), or erythropoietin (EPO) for growth and survival. We isolated a novel subline, UT-7/GM after long-term culture of UT-7 with GM-CSF. The hemoglobin concentration and γ-globin and EPO-receptor mRNA levels were significantly higher in EPO-treated UT-7/GM cells than in untreated cells. In contrast, the platelet factor 4 and glycoprotein IIb mRNA levels were much higher in thrombopoietin (TPO)-treated UT-7/GM cells than in untreated cells. Some TPO-treated cells had morphologically mature megakaryocytic characteristics such as a developed demarcation membrane in the cytoplasm and multilobular nuclei. These findings indicate that UT-7/GM is a bipotential cell line that can be induced to differentiate into erythroid and megakaryocytic lineages by EPO and TPO, respectively. Moreover, a minority of UT-7/GM cells acquired a high hemoglobin concentration by treatment with TPO, suggesting that TPO in part induced the erythroid differentiation of the UT-7/GM cells. Interestingly, GM-CSF inhibited the EPO- or TPO-induced erythroid differentiation and the TPO-induced megakaryocytic differentiation of UT-7/GM cells. These results support the hypothesis that cytokines influence the programming of gene expression required for lineage commitment or differentiation.

Targeting BCR‐ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and erythropoietin (EPO) on TF‐1/bcr‐abl (which was generated by transduction of a bcr‐abl fusion gene into the TF‐1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF‐1/bcr‐abl cells but not in the parental TF‐1 cells. However, GM‐CSF, a survival factor of the parental TF‐1 cells, protected TF‐1/bcr‐abl cells from imatinib‐induced apoptosis in a dose‐dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM‐CSF, accompanied by upregulation of Bcl‐xL and downregulation of p27/Kip1. In addition, although untreated TF‐1/bcr‐abl cells had lost responsiveness to both GM‐CSF and EPO and showed autonomous growth, GM‐CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib‐treated TF‐1/bcr‐abl cells differentiated into hemoglobin‐positive cells in the presence of EPO, as in the case for the parental TF‐1 cells. Taken together, imatinib‐treated CML cells may differentiate into mature cells in the presence of differentiation‐inducing cytokines such as EPO.